The establishment of cell polarity is a fundamental process in eukaryotic cell biology, and is often disrupted in human diseases including cancer. In animals, the PAR protein system mediates polarization of a wide variety of cell types, but how PAR proteins function at the molecular level to polarize cells is not well understood. Here, I propose an innovative approach to determine how cell polarity is established by regulated biochemical interactions between proteins in the PAR system, using the polarization of the 1-cell C. elegans embryo as a model system. My career goal is to establish an independent research program dedicated to the study of cell polarity in animals. As a first step to achieving this goal,I have pursued postdoctoral training in C. elegans developmental biology and genetics, complementing my background in biochemistry and cell biology. The C. elegans embryo is an excellent model for the study of cell polarity and morphogenesis due to its optical clarity and highly stereotyped developmental program. To further enable my research goals, I developed new methods for genome editing and single-embryo biochemistry in this experimental system. With these tools in hand, I can now use C. elegans to elucidate, for the first time, the molecular mechanism of cell polarization by the PAR protein system in vivo. Here I propose to: 1) Identify regulated PAR protein interactions required for cell polarization; 2) Use modeling and targeted experiments to determine how these interactions lead to stable cell polarity; and 3) Determine how the PAR system responds to cellular cues that act during polarity establishment. Completion of these aims will represent a major step forward in our understanding of the PAR protein system, and will lay the groundwork for my long term goals understanding how cell polarity pathways are integrated with other cellular process during morphogenesis. To achieve my aims, I will need additional training in the areas of microfluidics (advisory committee member Dr. Nancy Allbritton), microscopy and image analysis (advisory committee member Dr. Paul Maddox), and mathematical and computational modeling (advisory committee member Dr. Ed Munro), along with ongoing training in the cell and developmental biology of C. elegans from my mentor, Dr. Bob Goldstein. A K99/R00 award will allow me to establish an innovative research program that will make me a strong candidate for an independent position at a leading U.S. research institution. My work will provide important insights into the function of PAR proteins, which are fundamental players in the structure and function of many human and animal cell types.